This invention relates to an analytical element, particularly an analytical element for analyzing predetermined specific components in a fluid. More specifically, it relates to an analytical element for detecting and quantitatively analyzing .alpha.-amylase in a biological fluid sample.
Heretofore, many methods for analyzing a specific component in a fluid sample have been developed. These methods can be roughly classified into a reaction system (wet chemistry) where a reaction is carried out in a solution, and another reaction system (dry chemistry) where a reaction is carried out in a solid phase carrier. They have problems in operating characteristics or measuring accuracy.
Besides the conventional methods mentioned above, there are known multi-layer analytical elements in which the dry chemistry easy to operate is employed and which has a quantitative character. Such multi-layer analytical elements are disclosed in, for example, Japanese Patent Publication No. 21677/1978, and Japanese Provisional Patent Publications No. 164356/1980, No. 125847/1982, No. 197466/1982 and No. 90167/1983.
In the elements disclosed therein, all the reagents necessary for an analytical reaction are incorporated into one analytical element, and the concentration of a specific substance in a sample can be determined as follows: A certain amount of a serum or a whole blood is dropped onto the above-mentioned element; the latter is maintained at a selected temperature for a certain period of time; the determination of a reflected concentration is then carried out from a support side of the element; and on the basis of the reflected concentration, the concentration of the specific material is finally determined.
The aforesaid method has a higher analytical accuracy as compared with a conventional test paper type element, and possesses a reliability equal to or higher than the wet chemistry without needing to previously prepare any reagent.
The analytical element for analyzing .alpha.-amylase which has such characterictics is disclosed in Japanese Provisional Patent Publication No. 131089/1978. The disclosed analytical element comprises a light-transmissive and liquid-impervious support, and, for example, a registration layer, a blocking layer and a diffusion-reagent layer mentioned above is composed of an insoluble starch chemically bonded to a dye and an isotropically porous matrix; the blocking layer is composed of light-reflective titanium dioxide and a hydrophilic binder; and the registration layer is composed of a hydrophilic binder and a mordant.
According to the above-mentioned constitution, .alpha.-amylase in the fluid sample will break an .alpha.-1,4-bond of the dye-bonded insoluble starch in the diffusion-reagent layer, and the resulting diffusive fragments of the dye-bonding insoluble starch will permeate through the blocking layer and will bond to the mordant of the registration layer in order to give a dye.
In such an analytical element for analyzing .alpha.-amylase, there is utilized a so-called dystuff-bonded starch method (a chromogenic method) which is a kind of solution process. Originally, the dyestuff-bonded starch method comprises causing .alpha.-amylase in a specimen material to act on the insoluble dyestuff-bonding starch which is a substrate; filtering resultant fragments of a soluble dyestuff-bonding starch having a low-molecular weight; and carrying out a colorimetry to determine an activity of the .alpha.-amylase. It is definite that in this method, the separation between the unreacted insoluble dyestuff-bonding starch and the soluble dyestuff-bonding starch which is the reaction product is important.
In the above-mentioned analytical element for .alpha.-amylase, this separation depends on the diffusion of the fragments of the soluble dyestuff-bonding starch, which is the reaction product, from the diffusion-reagent layer to the registration layer through the blocking layer and the mordanting in the registration layer, but the diffusion of such a macromolecule is greatly affected by components in a specimen material. For this reason, the above-mentioned analytical element has disadvantages such as the formation of an non-uniform color density and the occurence of rings due to a chromatic phenomenon, and it is thus considered that the analytical element just described is noticeably poor in accuracy. Further, since the unreacted substrate and the reaction product have the same dyestuff, the blocking layer for blocking the color of the unreacted substrate must be disposed between the registration layer and the diffusion-reagent layer in order to distinguish these two colors.
In Japanese Provisional Patent Publication No. 40649/1982there is disclosed other analytical element for .alpha.-amylase. This analytical element comprises a light-transmissive and liquid-impervious support, and a second reagent layer and a first reagent layer provided in turn on the support. The first reagent layer above includes an undiffusible starch having dyestuff-forming reactive group, and the second reagent layer contains a chromogen compound.
According to the above-mentioned analytical element having such a constitution, the undiffusible starch having dyestuff-forming reactive group will be decomposed to change into a lower-molecular diffusible starch having dyestuff-forming reactive group by the .alpha.-amylase in a fluid sample, and the diffusible starch will permeate into the second layer and will react with the chromogen compound in order to form a dyestuff. For the formed dyestuff, a colorimetry will then be carried out to quantitatively measure the .alpha.-amylase.
The substrate having the dyestuff-forming reactive groups which is present in the first reagent layer is insoluble, therefore it will not permeate into the second reagent layer. In consequence, the unreacted substrate will not react with the undiffusible chromogen compound present in the second reagent layer so as to develop a color. Accordingly, this analytical element advantageously needs no blocking layer particularly which is essential in the analytical element for .alpha.-amylase disclosed in Japanese Provisional Patent Publication No. 131089/1978 referred to hereinbefore.
However, the analytical element for .alpha.-amylase disclosed in Japanese Provisional Patent Publication No. 40649/1982 is constitutionally the same as the analytical element disclosed in Japanese Provisional Patent Publication No. 131089/1978 in that the undiffusible substrate contained in the first reagent layer will be decomposed to change into the diffusible reaction product by the action of the .alpha.-amylase and the resulting reaction product will permeate into the second reagent layer. The aforesaid reaction product comprises macromolecules including several or dozens of glucose units, though being diffusible. Therefore, its diffusion from the first reagent layer to the second reagent layer will be, needless to say, non-uniform. In addition thereto, the reaction of the dyestuff-forming reactive groups in the reaction product of the substrate with the chromogen compound in the second reagent layer will be carried out in the noticeably scattering manner. It is thus fair to say that the aforementioned analytical element for .alpha.-amylase is remarkably poor in accuracy.
The .alpha.-amylase analysis based on the solution method in which a starch-iodine reaction process is employed is simple and convenient, but it has faults such as the deterioration of the starch by microorganism and the sublimation of the iodine, and also when it is used, much attention must be paid to the handling of an iodine solution which is corrosive.
Thus, the development of multi-layer analytical elements is now desired which are easy to operate and which are high in the reliability of a measuring accuracy and the like.